Field of the Invention
The present invention relates to an efficient method for assaying nucleic acids and proteins by fluorescence.
Description of the Related Arts
The medical and biological fields have increasingly employed the methods for detecting the nucleic acids. Such method have been used in various fields, i.e., clinical medicine field for hereditary disease diagnosis and virus inspection; breeding field for breeding qualification, and seeds appraisal; and forensic medicine field for determining paternity and criminal investigation.
Such assaying methods, in general utilize a labeled probe, for example, a DNA (or RNA) probe which is labeled with a radioactive isotope, hybridizing the labeled probe with a target nucleic acid and then detecting the target nucleic acid by autoradiography
However, hybridization methods which utilize radioactive isotypes comprise many drawbacks which pose serious obstacles to further application and development of this technology.
The drawbacks of hybridization methods which use radioactive isotope labels are as follows.
(a) In nucleic acid hybridization, the method lacks any spatial resolution sufficient to reveal relative positional relationship between contiguous signal. PA1 (b) Experimental procedures using isotope can be only carried out in isotope laboratories equipped with special facilities. This is a cause for hindering application of the hybridization method especially for clinical diagnosis. PA1 (c) Use of isotope is dangerous for laboratory workers even in laboratories. In addition, a danger for ordinary people always exists because of wastes, and the like. PA1 (d) A long time (several weeks to several months) may be required for detection, so the use of such methods for rapid clinical diagnosis is difficult. PA1 (e) Radioactivity decays with a definite half-life period so that experiments should be scheduled to fit a purchase date of isotope. If the schedule chart is slightly altered, there would be a danger of wasting isotope or experimental results in a large scale. PA1 (f) In order to enhance detection sensitivity, it is required to incorporate radioactivity to a nucleic acid probe as high as possible. However, the nucleic acid labeled enough to increase its radioactivity easily suffers from radioactive disintegration. PA1 (g) In general, radioactive isotypes are extremely expensive. This prevents further use of such hybridization methods.
In view of such background, some DNA or RNA labeling method in place of radioactive isotope have been developed.
For example, Japan Patent Application Laid Open No. 215300/1989 discloses Digoxigenin labeling method developed by the company named Boehringer Mannheim. This method employs alkaline phosphatase as enzyme, and BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium chloride) as its substrate. PCT Patent WO 88/00695 discloses the use of AMPPD (4-Methoxy-4-(3-phosphatephenyl) spiro(1,2-dioxetane-3,2'-adamantane) developed by Tropix Co., as the substrate of the alkaline phosphatase. This method with the aid of chemical emission has contributed to reduce the detection period.
Both sensitivities of the above two methods are lower than that of RI (radio isotope) labeling method. However, they are sufficiently high to facilitate the use of such methods.
In the former method (hereinafter referred to as BCIP/NBT method) for detecting the weak signal by color development, it is difficult to take a picture of the signal to be stored as clear data. Since the signals on a nylon membrane filter gets discolored over time, they may not last six months at longest. In the above aspect, this method is not suitable for data storage.
The latter method (hereinafter referred to as AMPPD method) overcomes the disadvantage of BCIP/NBT method as described above, i.e., data storage, and contributes to reduce the detection period. The resolution of this method, however, is substantially inferior to that of the former method. In instances wherein the detected signal amounts varies in a wide range, plural samples should be prepared by executing detection two or more times with each condition varied for synthetic judgment. Such work requires well-experienced technique and sufficient time. Since this method employs X-ray film, the result cannot be obtained until it is photosensitized, resulting in poor operability.